Method of transplanting cells by contacting donor cells with B7-1-and B7-2-specific immunoglobulins

ABSTRACT

The invention relates to a humanized anti-B7-2 antibody that comprises a variable region of nonhuman origin and at least a portion of an immunoglobulin of human origin. The invention also pertains to methods of treatment for various autoimmune diseases, transplant rejection, inflammatory disorders and infectious diseases by administering humanized anti-B7-2 and/or anti-B7-1 antibodies.

RELATED APPLICATION(S)

This application is a divisional of U.S. application Ser. No. 09/249,011filed Feb. 12, 1999, the entire teachings of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Antigen specific T-cell activation and the initiation of an immuneresponse depend initially on the interaction of the T-cell receptor(TCR) complex with the peptide/major histocompatibility complex (MHC)present on antigen presenting cells (APC). B7 molecules, B7-1 and B7-2,are molecules which are present on APCs. A second “costimulatory”signal, provided by the interaction of B7-1 and B7-2 on the APC withtheir ligands CD28 and CTLA4 on T-cells, is required to complete T-cellactivation and the subsequent regulation of an immune response. A needexists to regulate the B7-1 and B7-2 pathway, referred to as theB7:cD28/CTLA4 pathway. A further need exists to develop treatments fordiseases that are affected by this pathway.

SUMMARY OF THE INVENTION

The invention relates to a humanized immunoglobulin having bindingspecificity for B7-2, wherein the immunoglobulin comprises an antigenbinding region of nonhuman origin (e.g. rodent) and at least a portionof human origin (e.g. a human constant region such as an IgG constantregion, a human framework region). In one embodiment, the human constantregion can also contain a mutation that reduces the effector function ofthe humanized immunoglobulin. In another embodiment, the humanizedimmunoglobulin, described herein, can compete with murine 3D1 forbinding to B7-2. In a particular embodiment, the antigen binding regionof the humanized immunoglobulin is derived from the 3D1 monoclonalantibody.

The humanized immunoglobulin having binding specificity for B7-2 cancomprise a constant region of human origin and an antigen bindingregion, wherein the antigen binding region of nonhuman origin comprisesone or more complementarity determining regions (CDRs) of rodent origin(e.g., derived from 3D1 monoclonal antibody) that binds to B7-2, and theportion of an immunoglobulin of human origin is derived from a humanframework region (FR). The antigen binding region can further comprise alight chain and a heavy chain, wherein the light and heavy chain eachhave three CDRs derived from the 3D1 antibody. The FR of the light chaincan be derived, for example, from the light chain of the human H2Fantibody and the heavy chain can be derived, for example, from the heavychain of the human I2R antibody. In a particular embodiment, theinvention is a humanized immunoglobulin having binding specificity forB7-2 that is derived from the cell line deposited with the American TypeCulture Collection (A.T.C.C.), Accession No. CRL-12524.

The invention also embodies a humanized immunoglobulin having a bindingspecificity for B7-2 comprising a heavy chain and/or a light chain. Thelight chain comprises a CDR (e.g., CDR1, CDR2 and CDR3) derived from anantibody of nonhuman origin which binds B7-2 and a FR derived from alight chain of human origin (e.g., H2F antibody). The heavy chaincomprises a CDR (e.g.,CDR1, CDR2 and CDR3) derived from an antibody ofnonhuman origin which binds B7-2 and a FR region derived from a heavychain of human origin (e.g., the human III2R antibody). Theimmunoglobulin can further comprise CDR1, CDR2 and CDR3 for the light orheavy chain having the amino acid sequence set forth herein or an aminoacid.

One embodiment of the invention is a humanized-immunoglobulin lightchain having binding specificity for B7-2 comprising CDR1, CDR2 and/orCDR3 of the light chain of murine 3D1 antibody, and a human light chainFR (e.g., H2F antibody). Another embodiment is a humanizedimmunoglobulin light chain that comprises a variable region shown inFIG. 2B (SEQ ID NO: 8). The invention also relates to an isolatednucleic acid sequence that encodes a humanized variable light chainspecific for B7-2 that comprises a nucleic acid, such as the sequenceshown in FIG. 2B (SEQ ID NO: 7), a nucleic acid that encodes the aminoacid sequence shown in FIG. 2B (SEQ ID NO: 8), a nucleic acid whichhybridizes thereto under stringent hybridization conditions, and anucleic acid which is the complement thereof.

Another embodiment of the invention is a humanized immunoglobulin heavychain that is specific for B7-2 and comprises CDR1, CDR2 and/or CDR3 ofthe heavy chain of the 3D1 antibody, and a human heavy chain FR (e.g.,III2R antibody). The invention pertains to a humanized immunoglobulinheavy chain that comprises a variable region shown in FIG. 2A (SEQ IDNO: 6). The invention also pertains to an isolated nucleic acid sequencethat encodes a humanized variable heavy chain specific for B7-2 thatcomprises a nucleic acid, such as the sequence shown in FIG. 2A (SEQ IDNO: 5), a nucleic acid that encodes the amino acid sequence shown inFIG. 2A (SEQ ID NO: 6), a nucleic acid which hybridizes thereto understringent hybridization conditions, and a nucleic acid which is thecomplement thereof.

In particular, an embodiment of the invention is a humanizedimmunoglobulin which specifically binds to B7-2 and comprises ahumanized light chain comprising three light chain CDRs from the mouse3D 1 antibody and a light chain variable region framework sequence froma human immunoglobulin light chain, and a humanized heavy chaincomprising three heavy chain CDRs from the mouse 3D 1 antibody and aheavy chain variable region framework sequence from a humanimmunoglobulin heavy chain. The mouse 3D1 antibody can further have amature light chain variable domain, such as the mature light chainvariable domain shown in FIG. 1B (SEQ ID NO.: 4) and a mature heavychain variable domain such as the mature heavy chain variable regionshown in FIG. 1A (SEQ ID NO.: 2).

The invention includes an expression vector that comprises a fused genewhich encodes a humanized immunoglobulin light and/or heavy chain. Thegene comprises a nucleotide sequence encoding a CDR derived from a lightand/or heavy chain of a nonhuman antibody having binding specificity forB7-2 (e.g., murine 3D1 antibody) and a FR derived from a light and/orheavy chain of human origin.

The present invention also relates to a host cell comprising a nucleicacid of the present invention, including one or more constructscomprising nucleic acid of the present invention. In one embodiment, theinvention encompasses a host cell comprising a first recombinant nucleicacid that encodes a humanized immunoglobulin light chain and a secondrecombinant nucleic acid that encodes a humanized immunoglobulin heavychain. The first nucleic acid comprises a nucleotide sequence encoding aCDR derived from the light chain of murine 3D1 antibody and a FR derivedfrom a light chain of human origin. The second nucleic acid comprises anucleotide sequence encoding a CDR derived from the heavy chain ofmurine 3D 1 antibody and a FR derived from a heavy chain of humanorigin. The invention further relates to a host cell comprising a vectoror a nucleic acid that encodes the humanized immunoglobulin, asdescribed herein.

The invention further pertains to methods of preparing a humanizedimmunoglobulin that comprise maintaining a host cell that encodes ahumanized immunoglobulin that is specific for B7-2, as described herein,under conditions appropriate for expression of a humanizedimmunoglobulin, wherein a humanized immunoglobulin chain (one or more)are expressed and a humanized immunoglobulin is produced. The methodfurther comprises the step of isolating the humanized immunoglobulin.

Additional methods encompassed by the invention include a method ofinhibiting the interaction of a first cell bearing a B7-2 receptor witha second cell bearing B7-2, comprising contacting the second cell withan effective amount of a humanized immunoglobulin, as described herein.Accordingly, the invention relates to various methods of treatment. Theinvention includes a method for modulating an immune response of apatient or treating a patient having a transplanted organ, tissue, cellor the like comprising administering an effective amount of thehumanized immunoglobulin, as described herein, in a carrier (e.g.,pharmaceutical carrier), wherein the immune response is modulated. Theinvention pertains to treating acute and/or chronic transplant rejectionfor a prolonged periods of time (e.g., days, months, years). Theinvention also pertain to methods of treating a disease associated withmodulation of the B7-2 molecule (e.g., autoimmune diseases, infectiousdiseases, inflammatory disorders, systemic lupus erythematosus, diabetesmellitus, insulitis, arthritis, inflammatory bowel disease, inflammatorydermatitis, and multiple sclerosis), comprising administering to apatient an effective amount (e.g., a therapeutically effective amount)of a humanized immunoglobulin, as described herein, in a carrier.Accordingly, the invention encompasses a pharmaceutical compositioncomprising the humanized antibody, as described herein.

The invention also embodies a method of making a humanizedimmunoglobulin specific to B7-2 from a murine antibody specific to B7-2.The method comprises determining the CDRs of an antibody of non-humanorigin (e.g., murine origin) which has binding specificity for B7-2;obtaining a human antibody having a framework region amino acid sequencesuitable for grafting of the CDRs, and grafting the CDRs of an antibodyof non-human origin into the FR of the human antibody.

The invention also relates to a method for determining the presence orabsence of B7-2 in a sample. The method comprises obtaining the sampleto be tested, contacting the sample with a humanized antibody specificto B7-2, or a fragment thereof, sufficiently to allow formation of acomplex between B7-2 and the anti-B7-2 antibody, and detecting thepresence or absence of the complex formation. The presence of thecomplex indicates the presence of B7-2 in the sample.

The invention relates to methods for treating a patient having a diseasecomprising administering a therapeutically effective amount of ahumanized immunoglobulin specific to B7-1 and a therapeuticallyeffective amount of a humanized immunoglobulin specific to B7-2. Thediseases, as described herein, include, for example, autoimmunediseases, infectious diseases, asthma, inflammatory disorders, systemiclupus erythematosus, diabetes mellitus, insulitis, arthritis,inflammatory bowel disease, inflammatory dermatitis, and multiplesclerosis. This method also pertains to modulating the immune responseof a patient having a transplanted organ, tissue, cell or the likecomprising administering an effective amount of a humanizedimmunoglobulin that binds to B7-1 and a humanized immunoglobulin thatbinds to B7-2. Such diseases are described herein.

The invention also pertains to methods for transplanting cells (e.g.,bone marrow, or blood cells or components) to a patient in need thereofcomprising obtaining cells (e.g., bone marrow, or blood cells orcomponents) from a donor, contacting the cells with an immunoglobulinspecific to B7-1, an immunoglobulin specific to B7-2 and recipientcells, thereby obtaining a mixture. The immunoglobulins and therecipient cells are maintained for a period of time sufficient fortolerance induction. The mixture (e.g., bone marrow or blood cellcomposition) is then introduced into the patient. The recipient cellscomprise a lymphocyte antigen (e.g. lymphocytes that express class 1antigens (MHCI) or peripheral blood lymphocyte (PBL)). Instead of usingrecipient cells, the method also comprise utilizing tissue, organs orcells that express MHC Class I antigens, B7-1 and/or B7-2 molecules. Thecells can be engineered to express recipient molecules. The cells fromthe donor can be bone marrow cells or cells/components from blood (e.g.,stem cells or immature cells). The B7 immunoglobulins are in contactwith the donor bone marrow and the recipient cells for a period of timethat is long enough to induce tolerance induction (e.g., about 1 to 48hours, and, preferably about 36 hours). A patient in need of such atransplant is one who has a disease that is benefitted by or treatablewith a bone marrow transplant. Such diseases, for example, areproliferative diseases (e.g. leukemia, lymphoma and cancer), anemia(e.g. sickle-cell anemia, thalassemia, and aplastic anemia) and myeloiddysplasia syndrome (MDS).

The invention includes methods for transplanting bone marrow to apatient having a disease (e.g., proliferative diseases such as leukemia,lymphoma, cancer; anemia (e.g., sickle-cell anemia, thalassemia, andaplastic anemia) and myeloid dysplasia syndrome that is treated with abone marrow transplant comprising obtaining bone marrow from a donor,and contacting the bone marrow with an immunoglobulin specific to B7-1and/or an immunoglobulin specific to B7-2 and recipient cells (e.g.,lymphocyte). The bone marrow, immunoglobulin(s) and recipient cells arein contact for a period of time sufficient for tolerance induction(e.g., about 1–48 hours, preferably about 36 hours). The method thencomprises re-introducing the treated bone marrow to the patient.

Advantages of the invention include the ability to regulate or modulatethe B7 costimulatory pathway. Manipulation of this costimulatory pathwaywith a humanized anti-B7-2 and/or anti-B7-1 antibody provides methods oftreatments for various diseases. The humanized B7-2 antibody maintainsabout the same specificity for B7-2 as the murine 3D1 antibody, but witha reduced immunogenicity in humans. Accordingly, the invention canadvantageously be used to treat immune-related diseases/disorders ordiseases in which the B7-2 molecule plays an important role.Particularly, the invention relates to methods for treating infectiousor autoimmune diseases and methods for modulating the immune responsefor patients with transplanted organs, tissue or cells.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing and other embodiments, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying figures.

FIG. 1A is a sequence listing illustrating the heavy chain variableregion nucleic acid and amino acid sequences (SEQ ID NOS: 1 and 2,respectively) of the murine 3D1 antibody, wherein the amino acidsequences of the CDRs (CDR1, CDR2 and CDR3) are underlined and the firstamino acids of the mature chains are double underlined.

FIG. 1B is a sequence listing illustrating the light chain variableregion nucleic acid and amino acid sequences (SEQ ID NOS: 3 and 4,respectively) of the murine 3D1 antibody wherein, the nucleic and aminoacid sequences of the CDRs (CDR1, CDR2 and CDR3) are underlined and thefirst amino acids of the mature chains are double underlined.

FIG. 2A is a sequence listing illustrating the heavy chain variableregion nucleic acid and amino acid sequences (SEQ ID NOs: 5 and 6,respectively) of the humanized 3D 1 antibody, wherein the nucleic andamino acid sequences of the CDRs (CDR1, CDR2 and CDR3) are underlinedand the first amino acids of the mature chains are double underlined.

FIG. 2B contains the light chain variable region nucleic acid and aminoacid sequences (SEQ ID NOs: 7 and 8, respectively) of the humanized 3D1antibody, wherein the nucleic and amino acid sequences of CDR1, CDR2 andCDR3. The CDRs are underlined and the first amino acids of the maturechains are double underlined.

FIG. 3 is a graph of competitive binding assays. The graph depicts theresults of a competitive binding assay of murine or humanized anti-humanB7-2 mAbs to CHO expressing rhB7-2 (CHO/hB7-2) on their surface.Increasing concentrations of unlabelled competitor antibodies wereincubated with CHO/hB7-2 cells in the presence of radiolabelled tracermurine anti-human B7-2 mAb and the ratio of bound/free antibody wasdetermined.

FIG. 4 is a graph depicting the results of a direct binding assay ofmurine or humanized anti-human B7-2 mAbs to CHO/hB7-2 cells. Increasingconcentrations of radiolabelled antibodies were incubated with CHO orCHO/hB7-2 cells and the amount of specific antibody bound to theCHO/hB7-2 cells was determined.

FIG. 5 is a graph depicting the results of a T cell proliferation assay.Increasing concentrations of murine or humanized anti-human B7-2 mAbswere added to CD28⁺ human T cells stimulated with PMA and CHO/hB7-2cells and the inhibition of T cell proliferation by these mAbs wasdetermined.

FIG. 6 is a graph depicting the results of a one way mixed lymphocytereaction (MLR) assay. Fixed concentrations of murine or humanizedanti-human B7-2 (IgG2.M3 isotype) or hCTLA41g were added to a mixture ofhuman responder and stimulator PBLs and the proliferation of theresponder PBLs was determined on days 3, 4, and 5 by the addition ofradiolabelled thymidine.

FIG. 7 is a graph depicting the results of a one way secondary MLR assayusing PBLs from a primary MLR as responders and PBLs from the same or adifferent individual as in the primary MLR as stimulators. The humanizedanti-human B7-2 mAb (IgG2.M3 isotype) was added to the primary MLR only.Proliferation of the responder PBLs in the secondary MLR was determinedon days 3, 4, and 5 by the addition of radiolabelled thymidine.

FIG. 8 is a graph depicting the results of a one way secondary MLR assayusing PBLs from a primary MLR as responders and PBLs from the same or adifferent individual as in the primary MLR as stimulators. The humanizedanti-human B7-1 and B7-2 mAbs (IgG2.M3 isotype) were added to theprimary MLR only. Proliferation of the responder PBLs in the secondaryMLR was determined on days 3, 4, and 5 by the addition of radiolabelledthymidine.

FIG. 9 is a graph depicting the anti-tetanus response in non-humanprimates immunized with tetanus toxoid. Cynomolgus monkeys wereimmunized with purified tetanus toxoid and treated with humanizedanti-B7-1 and humanized anti-B7-2 (IgG2.M3 isotype) antibodies. Serumanti-tetanus antibody titers (IgM & IgG) were measured weekly over a 12week period.

FIG. 10 is a graph showing the serum concentration of anti-B7-1 andanti-B7-2 (IgG2.M3 isotype) mAbs at various times after administrationof an I.V. dose of 10 mg/Kg.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a humanized immunoglobulin having bindingspecificity for B7-2, comprising an antigen binding region of nonhumanorigin and at least a portion of an immunoglobulin of human origin.Preferably, the humanized immunoglobulins can bind B7-2 with an affinityof at least about 10⁷M⁻¹, preferably at least about 10⁸M⁻¹, and morepreferably at least about 10⁹M⁻¹. In one embodiment, the humanizedimmunoglobulin includes an antigen binding region of nonhuman originwhich binds B7-2 and a constant region derived from a human constantregion. The human constant region can have non-human residues in theframework region. In another embodiment, the humanized immunoglobulinwhich binds B7-2 comprises a complementarity determining region (one ormore) of nonhuman origin and a variable framework region (one or more)of human origin, and optionally, a constant region of human origin.Optionally, the FR region of the immunoglobulin can comprise residues ofnon-human origin. For example, the humanized immunoglobulin can comprisea heavy chain and a light chain, wherein the light chain comprises acomplementarity determining region derived from an antibody of nonhumanorigin which binds B7-2 and a framework region derived from a lightchain of human origin, and the heavy chain comprises a complementaritydetermining region derived from an antibody of nonhuman origin whichbinds B7-2 and a framework region derived from a heavy chain of humanorigin. Also, the invention, individually or in a functionalcombination, embodies the light chain, the heavy chain, the variableregion, the variable light chain and the variable heavy chain.

The invention relates to a humanized B7-2 antibody that possessessubstantially the same binding specificity as the murine B7-2 antibody(e.g., 3D1) from which the humanized antibody is made, but with reducedimmunogenicity in primates (e.g., humans). The humanized B7-2 antibodyhas about a lessor, substantially the same, or greater binding affinityas the murine counterpart. See FIGS. 3 and 4.

Naturally occurring immunoglobulins have a common core structure inwhich two identical light chains (about 24 kD) and two identical heavychains (about 55 or 70 kD) form a tetramer. The amino-terminal portionof each chain is known as the variable (V) region, also referred to asthe “antigen binding” region, and can be distinguished from the moreconserved constant (C) regions of the remainder of each chain. Withinthe variable region of the light chain is a C-terminal portion known asthe J region. Within the variable region of the heavy chain, there is aD region in addition to the J region. Most of the amino acid sequencevariation in immunoglobulins is confined to three separate locations inthe V regions known as hypervariable regions or complementaritydetermining regions (CDRs) which are directly involved in antigenbinding. The variable region is the portion of the antibody that bindsto the antigen. The constant region allows for various functions such asthe ability to bind to Fc receptors on phagocytic cells, placentalcells, mast cells, etc. The light and heavy chains each have a variableregion and a constant region. Accordingly, the invention relates to ahumanized immunoglobulin having binding specificity to B7-2. Thehumanized immunoglobulin comprises a light chain and a heavy chain inwhich two light chains and two heavy chains form the tetramer.

The variable region further constitutes two types of regions, aframework region (FR) and a complementarity determining region (CDR).CDRs are hypervariable regions that contain most of the amino acidsequence variation in between immunoglobulins. Proceeding from theamino-terminus, these regions are designated CDR1, CDR2 and CDR3,respectively. See FIGS. 1A–1B and 2A–2B. The CDRs are connected by moreconserved FRs. Proceeding from the amino-terminus, these regions aredesignated FR1, FR2, FR3, and FR4, respectively. The locations of CDRand FR regions and a numbering system have been defined by Kabat et al.(Kabat, E. A. et al., Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, U.S.Government Printing Office (1991); Kabat, E. A. Structural Concepts inImmunology and Immunochemistry, Second Edition, Holt, Rinehart andWinston, N.Y. (1976); Kabat, E. A. Sequences of Immunoglobulin Chains:Tabulation and Analysis of Amino Acid Sequences of Precurrsors,V-regions, C-regions, J-Chain and β2-Microglobulins, U.S. Department ofHealth, Education and Welfare, Public Health Service, (1979); Kabat, E.A. Structural Concepts in Immunology and Immunochemistry, Holt, Rinehartand Winston, N.Y. (1968); Kabat, E. A. Experimental Immunochemistry,Second Edition, Springfield, Thomas (1967). During the process ofhumanizing an immunoglobulin, one or more of the CDRs from an antibodyhaving specificity for B7-2 from a non-human species is grafted into theFRs of a human antibody. In addition, certain non-human frameworksubstitutes can be made according to the methods described herein. Theresulting humanized antibody has CDRs from a non-human species such as amouse and FRs from a human antibody, whereby the humanized antibodymaintains its antigenic specificity and affinity to B7-2.

The invention also relates to a humanized immunoglobulin light chain ora humanized immunoglobulin heavy chain. In one embodiment, the inventionrelates to a humanized light chain comprising one or more light chainCDRs (e.g., CDR1 (SEQ ID NO: 16), CDR2 (SEQ ID NO: 18) and/or CDR3 (SEQID NO: 20)) of nonhuman origin and a human light chain framework region(See FIG. 2B). In another embodiment, the invention relates to ahumanized immunoglobulin heavy chain comprising one or more heavy chainCDRs (e.g., CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 12), and/or CDR3 (SEQID NO: 14)) of nonhuman origin and a human heavy chain framework region(See FIG. 2A). The CDRs can be derived from a nonhuman immunoglobulinsuch as murine heavy (e.g., SEQ ID NO: 1, FIG. 1A) and light (e.g., SEQID NO: 3, FIG. 1B) variable region chains which are specific to B7-2.

The invention also embodies the humanized anti-B7-2 antibody expressedby a cell line deposited with the A.T.C.C., 10801 University Boulevard,Manassas, Va. 02110-2209, on May 5, 1998, A.T.C.C. No: CRL-12524. Thecell line which expresses the humanized anti-B7-2 antibody, depositedwith the A.T.C.C., is designated as a recombinant CHO cell line(PA-CHO-DUKX-1538) expressing the humanized anti-human B7-2 (CD86)monoclonal antibody (#HF2-3D1) of the IgG2.M3 isotype.

Human immunoglobulins can be divided into classes and subclasses,depending on the isotype of the heavy chain. The classes include IgG,IgM, IgA, IgD and IgE, in which the heavy chains are of the gamma (γ),mu (μ), alpha (αc), delta (δ) or epsilon (ε) type, respectively.Subclasses include IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, in which theheavy chains are of the γ1, γ2, γ3, γ4, α1 and α2 type, respectively.Human immunoglobulin molecules of a selected class or subclass maycontain either a kappa (κ) or lambda (λ) light chain. See e.g., Cellularand Molecular Immunology, Wonsiewicz, M. J., Ed., Chapter 45, pp. 41–50,W.B. Saunders Co, Philadelphia, Pa. (1991); Nisonoff, A., Introductionto Molecular Immunology, 2nd Ed., Chapter 4, pp. 45–65, SinauerAssociates, Inc., Sunderland, Mass. (1984).

The terms “HF2.3D1” and “3D1” refer to a murine immunoglobulin specificto B7-2. The terms “humanized HF2.3D1,” “humanized 3D1” and “hu3D1”refer to a humanized immunoglobulin specific to B7-2.

The terms “immunoglobulin” or “antibody” include whole antibodies andbiologically functional fragments thereof. Such biologically functionalfragments retain at least one antigen binding function of acorresponding full-length antibody (e.g., for B7-2) and preferably,retain the ability to inhibit the interaction of B7-2 with one or moreof its receptors (e.g., CD28, CTLA-4). In a preferred embodiment,biologically functional fragments can inhibit binding of B7-2 formanipulation of the co-stimulatory pathway. Examples of biologicallyfunctional antibody fragments which can be used include fragmentscapable of binding to an B7-2, such as single chain antibodies, Fv, Fab,Fab′ and F(ab′)₂ fragments. Such fragments can be produced by enzymaticcleavage or by recombinant techniques. For instance, papain or pepsincleavage can be used to generate Fab or F(ab′)₂ fragments, respectively.Antibodies can also be produced in a variety of truncated forms usingantibody genes in which one or more stop codons have been introducedupstream of the natural stop site. For example, a chimeric gene encodingthe heavy chain of an F(ab′)₂ fragment can be designed to include DNAsequences encoding the CH, domain and hinge region of the heavy chain.The invention includes single chain antibodies (e.g., a single chain FV)that contain both portions of the heavy and light chains.

The term “humanized immunoglobulin” as used herein refers to animmunoglobulin comprising portions of immunoglobulins of differentorigin, wherein at least one portion is of human origin. For example,the humanized antibody can comprise portions derived from animmunoglobulin of nonhuman origin with the requisite specificity, suchas a mouse, and from immunoglobulin sequences of human origin (e.g.,chimeric immunoglobulin). These portions can be joined togetherchemically by conventional techniques (e.g., synthetic) or prepared as acontiguous polypeptide using genetic engineering techniques (e.g., DNAencoding the protein portions of the chimeric antibody can be expressedto produce a contiguous polypeptide chain). Another example of ahumanized immunoglobulin of the invention is an immunoglobulincontaining one or more immunoglobulin chains comprising a CDR derivedfrom an antibody of nonhuman origin and a framework region derived froma light and/or heavy chain of human origin (e.g., CDR-grafted antibodieswith or without framework changes). Chimeric or CDR-grafted single chainantibodies are also encompassed by the term humanized immunoglobulin.See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al.,European Patent No. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397;Boss et al., European Patent No. 0,120,694 B1; Neuberger, M. S. et al.,WO 86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276 B1;Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400B1; Padlan, E. A. et al., European Patent Application No. 0,519,596 A1.See also, Ladner et al., U.S. Pat. No. 4,946,778; Huston, U.S. Pat. No.5,476,786; and Bird, R. E. et al., Science, 242: 423–426 (1988)),regarding single chain antibodies.

As embodied in the exemplified antibody of the present invention, theterm “humanized immunoglobulin” also refers to an immunoglobulincomprising a human framework, at least one CDR from a non-humanantibody, and in which any constant region present is substantiallyidentical to a human immunoglobulin constant region, e.g., at leastabout 60–90%, preferably at least 95% identical. Hence, all parts of ahumanized immunoglobulin, except possibly the CDR's, are substantiallyidentical to corresponding parts of one or more native humanimmunoglobulin sequences. In some instances, the humanizedimmunoglobulin, in addition to CDRs from a non-human antibody, wouldinclude additional non-human residues in the human framework region.

The design of humanized immunoglobulins can be carried out as follows.When an amino acid falls under the following categories, the frameworkamino acid of a human immunoglobulin to be used (acceptorimmunoglobulin) is replaced by a framework amino acid from aCDR-providing non-human immunoglobulin (donor immunoglobulin):

-   -   (a) the amino acid in the human framework region of the acceptor        immunoglobulin is unusual for human immunoglobulin at that        position, whereas the corresponding amino acid in the donor        immunoglobulin is typical for human immunoglobulin in that        position:    -   (b) the position of the amino acid is immediately adjacent to        one of the CDR's; or    -   (c) the amino acid is capable of interacting with the CDR's in a        tertiary structure immunoglobulin model (see, Queen et al., op.        cit., and Co et al., Proc. Natl. Acad. Sci. USA 88, 2869        (1991)).

For a detailed description of the production of humanizedimmunoglobulins, See Queen et al., op. cit. and Co et al, op. cit. andU.S. Pat. Nos. 5,585,089; 5,693,762, 5,693,761, and 5,530,101.

Usually, the CDR regions in humanized antibodies are substantiallyidentical, and more usually, identical to the corresponding CDR regionsin the mouse antibody from which they were derived. Although not usuallydesirable, it is sometimes possible to make one or more conservativeamino acid substitutions of CDR residues without appreciably affectingthe binding affinity of the resulting humanized immunoglobulin.Occasionally, substitutions of CDR regions can enhance binding affinity.

Other than for the specific amino acid substitutions discussed above,the framework regions of humanized immunoglobulins are usuallysubstantially identical, and more usually, identical to the frameworkregions of the human antibodies from which they were derived. Of course,many of the amino acids in the framework region make little or no directcontribution to the specificity or affinity of an antibody. Thus, manyindividual conservative substitutions of framework residues can betolerated without appreciable change of the specificity or affinity ofthe resulting humanized immunoglobulin.

The antigen binding region of the humanized immunoglobulin (thenon-human portion) can be derived from an immunoglobulin of nonhumanorigin, referred to as a donor immunoglobulin, having specificity forB7-2. For example, a suitable antigen binding region can be derived fromthe murine HF2.3D1 monoclonal antibody. U.S. Ser. No. 08/101,624, filedon Jul. 26, 1993, Ser. No. 08/109,393, filed Aug. 19, 1993 and08/147,773, filed Nov. 3, 1993, entitled, “B7-2:CTLA4/CD28 CounterReceptor”. See also, Freeman, et al., WO 95/03408, “B7-2: CTLA4/CD 28Counter Receptor, published on Feb. 2, 1995. Other sources includeB7-2-specific antibodies obtained from nonhuman sources, such as rodent(e.g., mouse and rat), rabbit, pig, goat or non-human primate (e.g.,monkey) or camelid animals (e.g., camels and llamas).

Additionally, other polyclonal or monoclonal antibodies, such asantibodies which bind to the same or similar epitope as the murineHF2.3D1 antibody, can be made (e.g., Kohler et al., Nature, 256:495–497(1975); Harlow et al., 1988, Antibodies: A Laboratory Manual, (ColdSpring Harbor, N.Y.); and Current Protocols in Molecular Biology, Vol. 2(Supplement 27, Summer '94), Ausubel et al., Eds. (John Wiley & Sons:New York, N.Y.), Chapter 11(1991)). For example, antibodies can beraised against an appropriate immunogen in a suitable mammal such as amouse, rat, rabbit, sheep, or camelid. Cells bearing B7-2, membranefractions containing B7-2, immunogenic fragments of B7-2, and a B7-2peptide conjugated to a suitable carrier are examples of suitableimmunogens (e.g., DNA or peptide immunogens). Antibody-producing cells(e.g., a lymphocyte) can be isolated, for example, from the lymph nodesor spleen of an immunized animal. The cells can then be fused to asuitable immortalized cell (e.g., a myeloma cell line), thereby forminga hybridoma. Fused cells can be isolated employing selective culturingtechniques. Cells which produce antibodies with the desired specificitycan be selected by a suitable assay, such as an ELISA. Immunoglobulinsof nonhuman origin having binding specificity for B7-2 can also beobtained from antibody libraries, such as a phage library comprisingnonhuman Fab molecules. Humanized immunoglobulins can be made usingmolecular biology techniques, Abgenics, or CAT techniques.

In one embodiment, the antigen binding region of the humanizedimmunoglobulin comprises a CDR of nonhuman origin. In this embodiment,the humanized immunoglobulin having binding specificity for B7-2comprises at least one CDR of nonhuman origin. For example, CDRs can bederived from the light and heavy chain variable regions ofimmunoglobulins of nonhuman origin, such that a humanized immunoglobulinincludes substantially the heavy chain CDR1 (e.g., SEQ ID NO: 10), CDR2(e.g., SEQ ID NO: 12) and/or CDR3 (e.g., SEQ ID NO: 14) amino acidsequences, and/or light chain CDR1 (e.g., SEQ ID NO: 16), CDR2 (e.g.,SEQ ID NO: 18) and/or CDR3 (e.g., SEQ ID NO: 20) amino acid sequences,from one or more immunoglobulins of nonhuman origin, and the resultinghumanized immunoglobulin has binding specificity for B7-2. All threeCDRs of a selected chain can be substantially the same as the CDRs ofthe corresponding chain of a donor, and preferably, all three CDRs ofthe light and heavy chains are substantially the same as the CDRs of thecorresponding donor chain. The nucleic acid sequences of the heavy chainCDR1 (e.g., SEQ ID NO: 9), CDR2 (e.g., SEQ ID NO: 11) and CDR3 (e.g.,SEQ ID NO: 13) and/or light chain CDR1 (e.g., SEQ ID NO: 15), CDR2(e.g., SEQ ID NO: 17), and CDR3 (e.g., SEQ ID NO: 19) can also be usedin grafting the CDRs into the human framework.

In another embodiment, the invention pertains to a humanizedimmunoglobulin having a binding specificity for B7-2 comprising a heavychain and a light chain. The light chain can comprise a CDR derived froman antibody of nonhuman origin which binds B7-2 and a FR derived from alight chain of human origin. For example, the light chain can compriseCDR1, CDR2 and/or CDR3 which have the amino acid sequence set forthbelow or an amino acid substantially the same as the amino acid sequencesuch that the antibody specifically binds to the B7-2: CDR1KSSQSLLNSRTRENYLA (SEQ ID NO: 16), CDR2 WASTRES (SEQ ID NO: 18), andCDR3 TQSYNLYT (SEQ ID NO: 20). The heavy chain can comprise a CDRderived from an antibody of nonhuman origin which binds B7-2 and a FRderived from a heavy chain of human origin. For example, the heavy chaincan comprise CDR1, CDR2 and CDR3 which have the amino acid sequence setforth below or an amino acid substantially the same as said amino acidsequence such that the antibody specifically binds to the B7-2: heavychain: CDR1 DYAIQ (SEQ ID NO: 10), CDR2 VINIYYDNTNYNQKFKG (SEQ ID NO:12), CDR3 AAWYMDY (SEQ ID NO: 14).

An embodiment of the invention is a humanized immunoglobulin whichspecifically binds to B7-2 and comprises a humanized light chaincomprising three light chain CDRs from the mouse 3D1 antibody and alight chain variable region framework sequence from a humanimmunoglobulin light chain. The invention further comprises a humanizedheavy chain that comprises three heavy chain CDRs from the mouse 3D1antibody and a heavy chain variable region framework sequence from ahuman immunoglobulin heavy chain. The mouse 3D1 antibody can furtherhave a mature light chain variable domain as shown in FIG. 1B (SEQ IDNO.: 4) and a mature heavy chain variable domain as shown in FIG. 1A(SEQ ID NO.: 2).

The portion of the humanized immunoglobulin or immunoglobulin chainwhich is of human origin (the human portion) can be derived from anysuitable human immunoglobulin or immunoglobulin chain. For example, ahuman constant region or portion thereof, if present, can be derivedfrom the κ or λ light chains, and/or the γ (e.g., γ1, γ2, γ3, γ4), μ, α(e.g., α1, α2), δ or ε heavy chains of human antibodies, includingallelic variants. A particular constant region, such as IgG2 or IgG4,variants or portions thereof can be selected to tailor effectorfunction. For example, a mutated constant region, also referred to as a“variant,” can be incorporated into a fusion protein to minimize bindingto Fc receptors and/or ability to fix complement (see e.g., Winter etal., U.S. Pat. Nos. 5,648,260 and 5,624,821; GB 2,209,757 B; Morrison etal., WO 89/07142; Morgan et al., WO 94/29351, Dec. 22, 1994). Inaddition, a mutated IgG2 Fc domain can be created that reduces themitogenic response, as compared to natural FC regions (see e.g., Tso etal., U.S. Pat. No. 5,834,597, the teachings of which are incorporated byreference herein in their entirety). See Example 3 for mutationsperformed to the humanized anti-B7-2 antibody.

If present, human FRs are preferably derived from a human antibodyvariable region having sequence similarity to the analogous orequivalent region of the antigen binding region donor. Other sources ofFRs for portions of human origin of a humanized immunoglobulin includehuman variable consensus sequences (See, Kettleborough, C. A. et al.,Protein Engineering 4:773–783 (1991); Queen et al., U.S. Pat. Nos.5,585,089, 5,693,762 and 5,693,761, the teachings all of which areincorporated by reference herein in their entirety). For example, thesequence of the antibody or variable region used to obtain the nonhumanportion can be compared to human sequences as described in Kabat, E. A.,et al., Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, U.S. Government PrintingOffice (1991). In a preferred embodiment, the FRs of a humanizedimmunoglobulin chain are derived from a human variable region having atleast about 60% overall sequence identity, and preferably at least about80% overall sequence identity, with the variable region of the nonhumandonor (e.g., murine HF2.3D1 antibody). For example, the overall sequenceidentity between the mouse HF2.3D1 and human H2F light chain variableframework regions is 82.5%, and the overall sequence identity betweenthe mouse HF2.3D1 and human I2R heavy chain variable framework regionsis 62.5%.

The phrase “substantially identical,” in context of two nucleic acids orpolypeptides (e.g., DNAs encoding a humanized immunoglobulin or theamino acid sequence of the humanized immunoglobulin) refers to two ormore sequences or subsequences that have at least about 80%, mostpreferably 90–95% or higher nucleotide or amino acid residue identity,when compared and aligned for maximum correspondence, as measured usingthe following sequence comparison method and/or by visual inspection.Such “substantially identical” sequences are typically considered to behomologous. Preferably, the “substantial identity” exists over a regionof the sequences that is at least about 50 residues in length, morepreferably over a region of at least about 100 residues, and mostpreferably the sequences are substantially identical over at least about150 residues, or over the full length of the two sequences to becompared. As described below, any two antibody sequences can only bealigned in one way, by using the numbering scheme in Kabat. Therefore,for antibodies, percent identity has a unique and well-defined meaning.

Amino acids from the variable regions of the mature heavy and lightchains of immunoglobulins are designated Hx and Lx respectively, where xis a number designating the position of an amino acid according to thescheme of Kabat, Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md., 1987 and 1991). Kabatlists many amino acid sequences for antibodies for each subgroup, andlists the most commonly occurring amino acid for each residue positionin that subgroup. Kabat uses a method for assigning a residue number toeach amino acid in a listed sequence, and this method for assigningresidue numbers has become standard in the field. Kabat's scheme isextendible to other antibodies not included in his compendium byaligning the antibody in question with one of the consensus sequences inKabat. The use of the Kabat numbering system readily identifies aminoacids at equivalent positions in different antibodies. For example, anamino acid at the L50 position of a human antibody occupies theequivalent position to an amino acid position L50 of a mouse antibody.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50–70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Thevariable regions of each light/heavy chain pair form the antibodybinding site. Thus, an intact antibody has two binding sites.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology, Paul, W., ed., 3rd ed. Raven Press, N.Y., 1993,Ch. 9).

From N-terminal to C-terminal, both light and heavy chain variableregions comprise alternating framework and (CDRs)” FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each region is inaccordance with the definitions of Kabat (1987) and (1991), supra and/orChothia & Lesk, J. Mol. Biol. 196:901–917 (1987); Chothia et al., Nature342:878–883 (1989).

In one embodiment, the humanized immunoglobulin comprises at least oneof the FRs derived from one or more chains of an antibody of humanorigin. Thus, the FR can include a FR1, FR2, FR3 and/or FR4 derived fromone or more antibodies of human origin. Preferably, the human portion ofa selected humanized chain includes FR1, FR2, FR3 and/or FR4 derivedfrom a variable region of human origin (e.g., from a humanimmunoglobulin chain, from a human consensus sequence). In a preferredembodiment, the FRs for the light chain variable region are from the H2Fhuman antibody and the FRs for the heavy chain variable region are fromthe I2R human antibody.

The immunoglobulin portions of nonhuman and human origin for use in theinvention have sequences that are identical to immunoglobulins orimmunoglobulin portions from which they are derived, or to variantsthereof. Such variants include mutants differing by the addition,deletion, or substitution of one or more residues. As indicated above,the CDRs which are of nonhuman origin are substantially the same as inthe nonhuman donor, and preferably are identical to the CDRs of thenonhuman donor. As described herein, changes in the FR, such as thosewhich substitute a residue of the FR of human origin with a residue fromthe corresponding position of the donor can be made. One or moremutations in the FR can be made, including deletions, insertions andsubstitutions of one or more amino acids. Several such substitutions aredescribed in the design of a humanized HF2.3D1 antibody in Example 2.For a selected humanized antibody or chain, framework mutations can bedesigned as described herein. Preferably, the humanized immunoglobulinscan bind B7-2 with an affinity similar to or better than that of thenonhuman donor. Variants can be produced by a variety of suitablemethods, including mutagenesis of nonhuman donor or acceptor humanchains.

The humanized immunoglobulins of the invention have binding specificityfor human B7-2, and include humanized immunoglobulins (includingfragments) which can bind determinants of B7-2. In a preferredembodiment, the humanized immunoglobulin of the present invention has atleast one functional characteristic of murine HF2.3D1 antibody, such asbinding function (e.g., having specificity for B7-2, having the same orsimilar epitopic specificity), and/or inhibitory function (e.g., theability to inhibit the binding of a cell bearing CD28 or CTLA-4 to theB7-2 ligand). Thus, preferred humanized immunoglobulins can have thebinding specificity of the murine HF2.3D 1 antibody, the epitopicspecificity of the murine HF2.3D1 antibody (e.g., can compete withmurine HF2.3D1, a chimeric HF2.3D1 antibody, or humanized HF2.3D1 forbinding to B7-2) and/or inhibitory function.

The binding function of a humanized immunoglobulin having bindingspecificity for B7-2 can be detected by standard immunological methods,for example using assays which monitor formation of a complex betweenhumanized immunoglobulin and B7-2 (e.g., a membrane fraction comprisingB7-2, or human lymphocyte cell line or recombinant host cell comprisingnucleic acid which expresses B7-2).

Binding and/or adhesion assays or other suitable methods can also beused in procedures for the identification and/or isolation of humanizedimmunoglobulins (e.g., from a library) with the requisite specificity(e.g., an assay which monitors adhesion between a cell bearing a B7-2receptor and B7-2, or other suitable methods).

The immunoglobulin portions of nonhuman and human origin for use in theinvention include light chains, heavy chains and portions of light andheavy chains. These immunoglobulin portions can be obtained or derivedfrom immunoglobulins (e.g., by de novo synthesis of a portion), ornucleic acids encoding an immunoglobulin or chain thereof having thedesired property (e.g., binds B7-2, sequence similarity) can be producedand expressed. Humanized immunoglobulins comprising the desired portions(e.g., antigen binding region, CDR, FR, C region) of human and nonhumanorigin can be produced using synthetic and/or recombinant nucleic acidsto prepare genes (e.g., cDNA) encoding the desired humanized chain. Toprepare a portion of a chain, one or more stop codons can be introducedat the desired position. For example, nucleic acid sequences coding fornewly designed humanized variable regions can be constructed using PCRmutagenesis methods to alter existing DNA sequences (see e.g., Kamman,M., et al., Nucl. Acids Res. 17:5404 (1989)). PCR primers coding for thenew CDRs can be hybridized to a DNA template of a previously humanizedvariable region which is based on the same, or a very similar, humanvariable region (Sato, K., et al., Cancer Research 53:851–856 (1993)).If a similar DNA sequence is not available for use as a template, anucleic acid comprising a sequence encoding a variable region sequencecan be constructed from synthetic oligonucleotides (see e.g., Kolbinger,F., Protein Engineering 8:971–980 (1993)). A sequence encoding a signalpeptide can also be incorporated into the nucleic acid (e.g., onsynthesis, upon insertion into a vector). If the natural signal peptidesequence is unavailable, a signal peptide sequence from another antibodycan be used (see, e.g., Kettleborough, C.A., Protein Engineering4:773–783 (1991)). Using these methods, methods described herein orother suitable methods, variants can be readily produced. In oneembodiment, cloned variable regions can be mutagenized, and sequencesencoding variants with the desired specificity can be selected (e.g.,from a phage library; see e.g., Krebber et al., U.S. Pat. No. 5,514,548;Hoogengoom et al., WO 93/06213, published Apr. 1, 1993)).

Nucleic Acids and Constructs Comprising Same:

The invention also relates to isolated and/or recombinant (including,e.g., essentially pure) nucleic acids comprising sequences which encodea humanized immunoglobulin or humanized immunoglobulin light or heavychain of the present invention.

Nucleic acids referred to herein as “isolated” are nucleic acids whichhave been separated away from the nucleic acids of the genomic DNA orcellular RNA of their source of origin (e.g., as it exists in cells orin a mixture of nucleic acids such as a library), and include nucleicacids obtained by methods described herein or other suitable methods,including essentially pure nucleic acids, nucleic acids produced bychemical synthesis, by combinations of biological and chemical methods,and recombinant nucleic acids which are isolated (see e.g., Daugherty,B. L. et al., Nucleic Acids Res., 19(9): 2471–2476 (1991); Lewis, A. P.and J. S. Crowe, Gene, 101: 297–302 (1991)).

Nucleic acids referred to herein as “recombinant” are nucleic acidswhich have been produced by recombinant DNA methodology, including thosenucleic acids that are generated by procedures which rely upon a methodof artificial recombination, such as the polymerase chain reaction (PCR)and/or cloning into a vector (e.g., plasmid) using restriction enzymes.“Recombinant” nucleic acids are also those that result fromrecombination events that occur through the natural mechanisms of cells,but are selected for after the introduction to the cells of nucleicacids designed to allow and make probable a desired recombination event.

The invention also relates more specifically to isolated and/orrecombinant nucleic acids comprising a nucleotide sequence which encodesa humanized HF2.3D1 immunoglobulin, also referred to as humanized 3D1(e.g., a humanized immunoglobulin of the invention in which the nonhumanportion is derived from the murine HF2.3D1 monoclonal antibody) or chainthereof. In one embodiment, the light chain comprises threecomplementarity determining regions derived from the light chain of theHF2.3D1 antibody, and the heavy chain comprises three complementaritydetermining regions derived from the heavy chain of the HF2.3D 1antibody. Such nucleic acids include, for example, (a) a nucleic acidcomprising a sequence which encodes a polypeptide comprising the aminoacid sequence of the heavy chain variable region of a humanized HF2.3D1immunoglobulin (e.g., SEQ ID NO: 5, See FIG. 2A), (b) a nucleic acidcomprising a sequence which encodes a polypeptide comprising the aminoacid sequence of the light chain variable region of a humanized HF2.3D1immunoglobulin (e.g., SEQ ID NO: 7, See FIG. 2B), (c) a nucleic acidcomprising a sequence which encodes at least a functional portion of thelight or heavy chain variable region of a humanized HF2.3D1immunoglobulin (e.g., a portion sufficient for antigen binding of ahumanized immunoglobulin which comprises the chain). Due to thedegeneracy of the genetic code, a variety of nucleic acids can be madewhich encode a selected polypeptide. In one embodiment, the nucleic acidcomprises the nucleotide sequence of the variable region as set forth orsubstantially as set forth in FIG. 2A and/or FIG. 2B, including doubleor single-stranded polynucleotides. Isolated and/or recombinant nucleicacids meeting these criteria can comprise nucleic acids encodingsequences identical to sequences of humanized HF2.3D1 antibody orvariants thereof, as discussed above.

Nucleic acids of the invention can be used in the production ofhumanized immunoglobulins having binding specificity for B7-2. Forexample, a nucleic acid (e.g., DNA) encoding a humanized immunoglobulinof the invention can be incorporated into a suitable construct (e.g., avector) for further manipulation of sequences or for production of theencoded polypeptide in suitable host cells.

Method of Producing Humanized Immunoglobulins Having Specificity forB7-2:

Another aspect of the invention relates to a method of preparing ahumanized immunoglobulin which has binding specificity for B7-2. Thehumanized immunoglobulin can be obtained, for example, by the expressionof one or more recombinant nucleic acids encoding a humanizedimmunoglobulin having binding specificity for B7-2 in a suitable hostcell.

Constructs or expression vectors suitable for the expression of ahumanized immunoglobulin having binding specificity for B7-2 are alsoprovided. The constructs can be introduced into a suitable host cell,and cells which express a humanized immunoglobulin of the invention, canbe produced and maintained in culture. Suitable host cells can beprocaryotic, including bacterial cells such as E. coli, B. subtilis andor other suitable bacteria, or eucaryotic, such as fungal or yeast cells(e.g., Pichia pastoris, Aspergillus species, Saccharomyces cerevisiae,Schizosaccharomyces pombe, Neurospora crassa), or other lower eucaryoticcells, and cells of higher eucaryotes such as those from insects (e.g.,Sf9 insect cells (WO 94/26087, O'Connor, published Nov. 24, 1994)) ormammals (e.g., COS cells, NSO cells, SP2/0, Chinese hamster ovary cells(CHO), HuT 78 cells, 293 cells). (See, e.g., Ausubel, F. M. et al., eds.Current Protocols in Molecular Biology, Greene Publishing Associates andJohn Wiley & Sons Inc., (1993)).

Host cells which produce a humanized immunoglobulin having bindingspecificity for B7-2 can be produced as follows. For example, a nucleicacid encoding all or part of the coding sequence for the desiredhumanized immunoglobulin can be inserted into a nucleic acid vector,e.g., a DNA vector, such as a plasmid, virus or other suitableexpression unit. A variety of vectors are available, including vectorswhich are maintained in single copy or multiple copy, or which becomeintegrated into the host cell chromosome.

Suitable expression vectors can contain a number of components,including, but not limited to one or more of the following: an origin ofreplication; a selectable marker gene; one or more expression controlelements, such as a transcriptional control element (e.g., a promoter,an enhancer, terminator), and/or one or more translation signals; asignal sequence or leader sequence for membrane targeting or secretion.In a construct, a signal sequence can be provided by the vector or othersource. For example, the transcriptional and/or translational signals ofan immunoglobulin can be used to direct expression.

A promoter can be provided for expression in a suitable host cell.Promoters can be constitutive or inducible. For example, a promoter canbe operably linked to a nucleic acid encoding a humanized immunoglobulinor immunoglobulin chain, such that it directs expression of the encodedpolypeptide. A variety of suitable promoters for procaryotic (e.g., lac,tac, T3, T7 promoters for E. coli) and eucaryotic (e.g., yeast alcoholdehydrogenase (ADH1, SV40, CMV) hosts are available.

In addition, the expression vectors typically comprise a selectablemarker for selection of host cells carrying the vector, and, in the caseof replicable expression vector, an origin or replication. Genesencoding products which confer antibiotic or drug resistance are commonselectable markers and may be used in procaryotic (e.g., β-lactamasegene (ampicillin resistance), Tet gene for tetracycline resistance) andeucaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolicacid), ampicillin, or hygromycin resistance genes). Dihydrofolatereductase marker genes permit selection with methotrexate in a varietyof hosts. Genes encoding the gene product of auxotrophic markers of thehost (e.g., LEU2, URA3, HIS3) are often used as selectable markers inyeast. Use of viral (e.g., baculovirus) or phage vectors, and vectorswhich are capable of integrating into the genome of the host cell, suchas retroviral vectors, are also contemplated. The invention also relatesto cells carrying these expression vectors.

For example, a nucleic acid (e.g., one or more nucleic acids) encodingthe heavy and light chains of a humanized immunoglobulin having bindingspecificity for B7-2, or a construct (e.g., one or more constructs)comprising such nucleic acid(s), can be introduced into a suitable hostcell by a method appropriate to the host cell selected (e.g.,transformation, transfection, electroporation, infection), such that thenucleic acid(s) are operably linked to one or more expression controlelements (e.g., in a vector, in a construct created by processes in thecell, integrated into the host cell genome). Host cells can bemaintained under conditions suitable for expression (e.g., in thepresence of inducer, suitable media supplemented with appropriate salts,growth factors, antibiotic, nutritional supplements, etc.), whereby theencoded polypeptide(s) are produced. If desired, the encoded protein(e.g., humanized HF2.3D1 antibody) can be isolated from (e.g., the hostcells, medium, milk). This process encompasses expression in a host cellof a transgenic animal (see e.g., WO 92/03918, GenPharm International,published Mar. 19, 1992).

Fusion proteins can be produced in which a humanized immunoglobulin orimmunoglobulin chain is linked to a non-immunoglobulin moiety (e.g., amoiety which does not occur in immunoglobulins as found in nature) in anN-terminal location, C-terminal location or internal to the fusionprotein. For example, some embodiments can be produced by the insertionof a nucleic acid encoding immunoglobulin sequences into a suitableexpression vector, such as a pET vector (e.g., pET-15b, Novagen), aphage vector (e.g., pCANTAB 5 E, Pharmacia), or other vector (e.g.,pRIT2T Protein A fusion vector, Pharmacia). The resulting construct canbe introduced into a suitable host cell for expression. Upon expression,some fusion proteins can be isolated or purified from a cell lysate bymeans of a suitable affinity matrix (see e.g., Current Protocols inMolecular Biology (Ausubel, F. M. et al., eds., Vol. 2, Suppl. 26, pp.16.4.1–16.7.8 (1991)).

Therapeutic Methods and Compositions:

Two types of T-cells exist: helper T cells and cytotoxic T cells. Thehelper T cells is a mechanism that can recognize the antigen when theantigen is coupled with a major histocompatibility complex (MHC).Antigen presenting cells internalize an antigen and re-express theantigen with the MHC molecule. Upon recognition of the antigen, asecretion of cytokines occur. Cytokine secretion activates B-lymphocytesand cytotoxic T cells, phagocytes and other cells. However, cytokinesecretion and cellular proliferation require more than recognition ofthe antigen. Complete T-cell activation requires a second signalreferred to as the “costimulatory signal.” These costimulatory signalsserve to initiate, maintain, and regulate the activation cascade. Animportant costimulatory pathway is called the B7:CD28/CTLA-4 pathway.

The B7:CD28/CTLA-4 pathway involves two costimulatory ligands, B7-1(CD80) and B7-2 (CD86). The B7-1 and B7-2 ligands which are present onthe antigen presenting cell each bind to two receptors on T-cells calledCD28 and CTLA-4.

The expression of B7 antigens, B7-1 (CD80) and B7-2 (CD86), is tightlyregulated. (Linsley, PS et al., Immunity 1:793–801 (1994). Unstimulatedantigen-presenting cells generally do not express B7-1 and B7-2, exceptin dendritic cells. After activation, dendritic and epidermalLangerhans' cells, B cells, and macrophages up-regulate the expressionof B7-2 and B7-1. Additionally, B7-2 can be expressed on granulocytesand on T-cell molecules, and B7-1 is expressed in fibroblasts and T-cellmolecules.

In most immune responses, B7-2 is induced earlier than B7-1 and rises tohigher levels. B7-2 also affects the production of interleukin-4 (IL-4)and the generation of type 2 helper cells. B7 molecules are alsoresponsible for costimulating CD8 T cells in the absence of CD4 T cellswhich can be helpful in generating vaccines against melanoma. B7molecules can costimulate natural killer cells and γ/δ T cells. Hence,modulation of B7 molecules is helpful in anti-tumor and anti-microbialimmunity.

The B7:CD28/CTLA-4 pathway participates in various disease statesincluding the pathogenesis of infectious diseases, asthma, autoimmunediseases, inflammatory disorders, the rejection of grafted organs andgraft versus host disease. This pathway also participates in prophylaxisand mechanisms that stimulate the immune system. Transfection with genesencoding costimulators, such as B7, are applicable for anti-tumor andanti-viral vaccines. Also, the B7 molecules participate in autoimmunediseases such as systemic lupus erythematosus, diabetes mellitus,insulitis, arthritis, inflammatory bowel disease, inflammatorydermatitis (psoriasis vulgaris and atopic dermatitis), and multiplesclerosis. Reiser, Hans, M. D., et al., Mechanisms of Disease, NewEngland J. of Med., Vol 335, No. 18, 1369 (1996).

Accordingly, the invention encompasses methods for treating the disease,as described herein, comprising administering immunoglobulin(s) thatbinds to B7-1 and/or B7-2. The immunoglobulin should be administered intherapeutically effective amounts and, optionally, in a carrier. Inaddition to the diseases described herein, the immunoglobulin that bindB7-1 and/or B7-2 can be administered to a person having transplantedtissue, organ or cells. Inhibiting the B7 pathway prevents or reducesthe rejection of the transplanted tissue, organ or cell. The inventionpertains to treating acute and/or chronic transplant rejection for aprolonged period of time (e.g., days, months, years).

Therefore, modulating or influencing the B7-2's role can be useful intreating patients with these diseases. B7-2 modulation is also useful intreating patients with immune-related or autoimmune diseases anddisorders in which B7-2 participates. The modulation of B7-2 can also beused for diseases related to or affected by IL-4 and/or the generationof type 2 helper cells. These disorders/diseases can be treated using anantibody specific to B7-2. Preferably, the antibody is a humanizedantibody specific to B7-2. Treatment of these diseases may befacilitated with co-administration of an anti-B7-2 antibody, includingchimeric and humanized versions thereof, with an anti-B7-1 antibody, orantibodies to the corresponding receptors, CD28 and CTLA-4. Methods oftreatment also involve co-administration of a humanized anti B7-2antibody or humanized anti B7-1 antibody with other standard therapydrugs, such methotrexate, cyclosporin, steroids, α CD40 ligands.

The invention includes methods for transplanting cells (e.g., bloodcells or components, or bone marrow) to a patient in need thereof. Apatient in need thereof is one, for example, having a disease that istreated with such a transplant (e.g., proliferative diseases such asleukemia, lymphoma, cancer), anemia such as sickle-cell anemia,thalassemia, and aplastic anemia) and myeloid dysplasia syndrome). Themethod comprises obtaining cells from a donor. Generally, donor bonemarrow contains both immature and mature lymphocytes. The blood cellsfrom a donor can be stem cells or immature blood cells in addition tobone marrow cells. The cells of the donor preferably comes from a personwho has similar characteristics as the patient/recipient (e.g., thedonor's bone marrow is a match to the patient's bone marrow). Thecharacteristics that are analyzed to determine whether a donor is amatch to the patient are MHC class 1 and 2 (e.g., HLA-A, HLA-B, and/orHLA-DR). The method involves contacting the cells (e.g., bone marrow orother blood components) with an immunoglobulin specific to B7-1 and/oran immunoglobulin specific to B7-2 and recipient cells (e.g., lymphocytefrom the patient) to obtain a mixture (e.g., treated cells). The donorcells, immunoglobulin(s) and recipient cells are in contact for a periodof time sufficient for tolerance induction (e.g., about 1–48 hours,preferably about 36 hours). Tolerance induction (e.g., anergy) refers tothe lack of responsiveness to an antigen that has been induced with atreatment with B7-1 and/or B7-2 antibodies, such that the T-cell can nolonger adequately or fully respond to that antigen. Example 9. Therecipient cells (e.g., Peripheral Blood Lymphocytes (PBL), orlymphocytes that express class I antigens (MHC-I)) are radiated toprevent cells from dividing. A substitute for recipient cells can betissue, organs or engineered cells that express MCH class I antigens,and B7-1 and/or B7-2 molecules. The method then includes introducing themixture (e.g., treated cells) or bone marrow to the patient. This methodof treatment is aimed at preventing graft vs. host disease. For example,cells in the treated bone marrow become tolerant to recipientalloantigen thereby reducing or eliminating graft vs. host disease.Accordingly, the claimed methods include treatment, preventing or aidingin the prevention of graft vs. host disease. The anti B7-1 and B7-2antibodies reduce rejection of the donor bone marrow. However, themethods are able to reduce rejection without significantly compromisingthe patient's ability to detect and develop an immune response to otherforeign cells and antigens. Hence, the methods allows thetransplantation to be recipient specific and reject foreign antigenswithout compromising the transplant. See Exemplification Section.

The terms “pharmaceutically acceptable carrier” or a “carrier” refer toany generally acceptable excipient or drug delivery device that isrelatively inert and non-toxic. A preferred embodiment is to administerthe immunoglobulin, (e.g., tablet or capsule form). Exemplary carriersinclude calcium carbonate, sucrose, dextrose, mannose, albumin, starch,cellulose, silica gel, polyethylene glycol (PEG), dried skim milk, riceflour, magnesium stearate, and the like. Suitable formulations andadditional carriers are described in Remington's PharmaceuticalSciences, (17th Ed., Mack Pub. Co., Easton, Pa.), the teachings of whichare incorporated herein by reference in their entirety.

Suitable carriers (e.g., pharmaceutical carriers) also include, but arenot limited to sterile water, salt solutions (such as Ringer'ssolution), alcohols, polyethylene glycols, gelatin, carbohydrates suchas lactose, amylose or starch, magnesium stearate, talc, silicic acid,viscous paraffin, fatty acid esters, hydroxymethylcellulose, polyvinylpyrolidone, etc. Such preparations can be sterilized and, if desired,mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, and/or aromatic substances and the likewhich do not deleteriously react with the immunoglobulin. They can alsobe combined where desired with other active substances, e.g., enzymeinhibitors, to reduce metabolic degradation. A carrier (e.g., apharmaceutically acceptable carrier) is preferred, but not necessary toadminister the immunoglobulin.

For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solutions, as well assuspensions, emulsions, or implants, including suppositories. Inparticular, carriers for parenteral administration include aqueoussolutions of dextrose, saline, pure water, ethanol, glycerol, propyleneglycol, peanut oil, sesame oil, polyoxyethylene-polyoxypropylene blockpolymers, and the like. Ampules are convenient unit dosages.

Immunoglobulins of the invention can be administered intravenously,parenterally, intramuscular, subcutaneously, orally, nasally, byinhalation, by implant, by injection, or by suppository. The compositioncan be administered in a single dose or in more than one dose over aperiod of time to confer the desired effect.

The actual effective amounts of immunoglobulin can vary according to thespecific immunoglobulin being utilized, the particular compositionformulated, the mode of administration and the age, weight and conditionof the patient, for example. As used herein, an effective amount of theimmunoglobulin is an amount which modulates or inhibits B7 molecules.Dosages for a particular patient can be determined by one of ordinaryskill in the art using conventional considerations, (e.g. by means of anappropriate, conventional pharmacological protocol).

The invention also pertains to methods for determining the presence,absence or level of B7-2 using a humanized anti-B7-2 antibody. Thepresence or absence of B7-2 can be detected in an assay (e.g., ELISA,radioimmunoassay (RIA) or FACS Immunohistochemistry). The assay can be adirect detection or an indirect detection (e.g. a competitive assay).

For example, to determine the presence or absence of B7-2 using an ELISAassay in a suitable sample, the method comprises combining a suitablesample with a composition comprising a humanized or murine anti B7-2antibody as detector (e.g., biotinylated anti B7-2 MAb andHRP-streptavidin, or HRP-conjugated anti-B7-2 Mab) and a solid support(e.g., a microtiter plate), having an anti-B7-2 capture antibody bound(directly or indirectly) thereto. The detector antibody can bind to adifferent B7-2 epitope from that recognized by the capture antibody,under conditions suitable for the formation of a complex between theanti-B7-2 antibodies and B7-2. The method further comprises determiningthe formation of complex in the sample.

The presence of B7-2 can also be determined in a radioimmunoassay. Forexample, the presence of B7-2 can be assessed by an immunobinding assaycomprising obtaining a sample, contacting the sample with a compositioncomprising an anti-B7-2 antibody (e.g., a humanized or murine anti-B7-2antibody comprising a radioactive label; or a humanized or murineanti-B7-2 antibody comprising a binding site for a second antibody whichcomprises a radioactive label), preferably in an amount in excess ofthat required to bind the B7-2, under conditions suitable for theformation of labeled complexes. The method further comprises determining(detecting or measuring) the formation of complex in the samples.

EXEMPLIFICATION

The present invention will now be illustrated by the following Examples,which are not intended to be limiting in any way.

Example 1 Cloning and Sequencing of Mouse 3D1 Variable Region cDNAs

Mouse 3D1 (also referred to as HF2.3D1) heavy and light chain variableregion cDNAs were cloned from mRNA isolated from hybridoma cells usinganchored PCR (Co et al., J. Immunol. 148: 1149 (1992)). The 5′ primersused annealed to the poly-dG tails added to the cDNA, and the 3′ primersannealed to the constant regions. The amplified gene fragments were theninserted into the plasmid pUC18. Nucleotide sequences were determinedfrom several independent clones for both V_(L) and V_(H) cDNA. For theheavy chain, a single, unique sequence was identified, typical of amouse heavy chain variable region. For the light chain, two uniquesequences, both homologous to murine light chain variable regionsequences, were identified. However, one sequence was not functionalbecause of a missing nucleotide that caused a frame shift at the V-Jjunction, and was identified as the non-productive allele. The othersequence was typical of a functional mouse kappa chain variable region.The variable region cDNA sequences of the heavy chain and the functionallight chain and the translated amino acid sequences are shown in FIGS.1A–1B. The mouse V_(L) sequence belongs to Kabat's mouse kappa chainsubgroup I. The mouse V_(H) belongs to Kabat's heavy chain subgroupII(A).

Example 2 Design of Humanized 3D1 Variable Regions

To retain the binding affinity of the mouse antibody in the humanizedantibody, the general procedures of Queen et al. were followed (Queen etal. Proc. Natl. Acad. Sci. USA 86:10029 (1989), U.S. Pat. Nos. 5,585,089and 5,693,762, the teachings of which are incorporated herein in theirentirety). The choice of framework residues can be critical in retaininghigh binding affinity. In principle, a framework sequence from any humanantibody can serve as the template for CDR grafting; however, it hasbeen demonstrated that straight CDR replacement into such a frameworkcan lead to significant loss of binding affinity to the antigen (Tempestet al., Biotechnology 9:266 (1992); Shalaby et al., J. Exp. Med. 17: 217(1992)). The more homologous a human antibody is to the original murineantibody, the less likely the human framework will introduce distortionsinto the mouse CDRs that could reduce affinity. Based on a sequencehomology, III2R was selected to provide the framework for the humanized3D1 heavy chain and H2F for the humanized 3D1 light chain variableregion. Manheimer-Lory, A. et al., J. Exp. Med. 174(6):1639–52 (1991).Other highly homologous human antibody chains would also be suitable toprovide the humanized antibody framework, especially kappa light chainsfrom human subgroup 4 and heavy chains from human subgroup 1 as definedby Kabat.

Normally the heavy chain and light chain from the same human antibodyare chosen to provide the framework sequences, so as to reduce thepossibility of incompatibility in the assembling of the two chains. TheIII2R antibody shows a high homology to the 3D1 heavy and light chainsand thus, was chosen to provide the framework for the initial humanizedantibody model. The 3D1 light chain variable region, however, shows asignificantly higher homology to the H2F framework compared to anyothers, including III2R. Therefore, H2F was chosen instead to providethe framework for the humanized 3D1 light chain variable region, whileIII2R was selected to provide the framework for the heavy chain variableregion.

The computer programs ABMOD and ENCODE (Levitt et al., J. Mol. Biol.168: 595 (1983)) were used to construct a molecular model of the 3D1variable domain, which was used to locate the amino acids in the 3D1framework that are close enough to the CDRs to potentially interact withthem. To design the humanized 3D1 heavy and light chain variableregions, the CDRs from the mouse 3D1 heavy chain were grafted into theframework regions of the human III2R heavy chain and the CDRs from themouse 3D1 light chain grafted into the framework regions of the humanH2F light chain. At framework positions where the computer modelsuggested significant contact with the CDRs, the amino acids from themouse antibody were substituted for the original human framework aminoacids. For humanized 3D1, this was done at residues 27, 30, 48, 67, 68,70 and 72 of the heavy chain and at residue 22 of the light chain.Furthermore, framework residues that occurred only rarely at theirpositions in the database of human antibodies were replaced by a humanconsensus amino acid at those positions. For humanized 3D1 this was doneat residue 113 of the heavy chain and at residue 3 of the light chain.

The sequence of the humanized 3D1 antibody heavy chain and light chainvariable regions is shown in FIGS. 2A–2B. However, many of the potentialCDR-contact residues are amenable to substitutions of other amino acidsthat may still allow the antibody to retain substantial affinity to theantigen. Table 1 lists a number of positions in the framework wherealternative amino acids may be suitable (LC=light chain, HU=heavychain). The position specified in the table are the number of aminoacids from the first amino acid of the mature chain, which is indicatedby a double underline (FIGS. 2A–2B). For example, position LC-22 is thetwenty second amino acid beginning from the doubled underlined AsparticAcid, D, (or the forty second amino acid from the start codon).

TABLE 1 Complementarity Determining Region Amino Acids Substitutesand/or Alternatives Position Humanized 3D1 Alternatives LC-22 S N HU-27Y G HU-30 T S HU-48 I M HU-67 K R HU-68 A V HU-70 M I HU-72 V A

Likewise, many of the framework residues not in contact with the CDRs inthe humanized 3D1 heavy and light chains can accommodate substitutionsof amino acids from the corresponding positions of III2R and H2Fframeworks, from other human antibodies, from the mouse 3D1 antibody, orfrom other mouse antibodies, without significant loss of the affinity ornon-immunogenicity of the humanized antibody. Table 2 lists a number ofadditional positions in the framework where alternative amino acids maybe suitable.

TABLE 2 Framework Region Amino Acid Substitutes and/or AlternativesPosition Humanized 3D1 Alternatives LC-3 V Q HU-113 T I

Selection of various alternative amino acids may be used to produceversions of humanized 3D1 that have varying combinations of affinity,specificity, non-immunogenicity, ease of manufacture, and otherdesirable properties. Thus, the examples in the above tables are offeredby way of illustration, not of limitation.

Example 3 Construction of Humanized 3D1

Once the humanized variable region amino acid sequences had beendesigned, as described above, genes were constructed to encode them,including signal peptides, splice donor signals and appropriaterestriction sites (FIGS. 2A–2B). The light and heavy chain variableregion genes were constructed and amplified using eight overlappingsynthetic oligonucleotides ranging in length from approximately 65 to 80bases (see He et al. J. Immunol. 160: 1029 (1998)). The oligos wereannealed pairwise and extended with the Klenow fragment of DNApolymerase I, yielding four double-stranded fragments. The resultingfragments were denatured, annealed, and extended with Klenow, yieldingtwo fragments. These fragments were denatured, annealed pairwise, andextended once again, yielding a full-length gene. The resulting productwas amplified by polymerase chain reaction (PCR) using Taq polymerase,gel-purified, digested with XbaI, gel-purified again, and subcloned intothe XbaI site of the pVk for the expression of light chain and pVg4 orpVg2.M3 for the expression of heavy chains. The pVk vector for kappalight chain expression has been previously described (See Co et al., J.Immunol. 148:1149 (1992)). The pVg4 vector for the gamma 4 heavy chainexpression was constructed by replacing the XbaI—BamHI fragment of pVg1containing the g1 constant region gene (See Co et al., J. Immunol. 148:1149 (1992)) with an approximately 2000 bp fragment of the human g4constant region gene (Ellison and Hood, Proc. Natl. Acad. Sci. USA79:1984 (1982)) that extended from the HindIII site preceding the C_(H)1exon of the g4 gene to 270 bp after the NsiI site following the C_(H)4exon of the gene. The pVg2.M3 vector for the gamma 2 heavy chainexpression was described in Cole, et al., J. Immunol. 159: 3613 (1997).The pVg2.M3 is mutated from the human wildtype IgG2 by replacing theamino acids Val and Gly at positions 234 and 237 with Ala. This varianthas a reduced interaction with its Fc receptors and thus has minimalantibody effector activity.

The structure of the final plasmids was verified by nucleotidesequencing and restriction mapping. All DNA manipulations were performedby standard methods well-known to those skilled in the art.

Two humanized 3D1, an IgG4 and an IgG2.M3, were generated forcomparative studies. To construct a cell line producing humanized 3D1(IgG4 or IgG2.M3), a light chain and the respective heavy chain plasmidswere transfected into the mouse myeloma cell line Sp2/0-Ag14 (ATCC CRL1581). Plasmids were also transfected into CHO cells using known methodsin the art. Before transfection, the heavy and light chain-containingplasmids were linearized using restriction endonucleases. The kappachain and the gamma2 chain were linearized using FspI; the gamma 4 chainwas linearized using BstZ17I. Approximately 20 μg of the light chain anda heavy chain plasmid was transfected into 1×10⁷ cells in PBS.Transfection was by electroporation using a Gene Pulser apparatus(BioRad) at 360 V and 25 μFD capacitance according to the manufacturer'sinstructions. The cells from each transfection were plated in four96-well tissue culture plates, and after two days, selection medium(DMEM, 10% FCS, 1xHT supplement (Sigma), 0.25 mg/ml xanthine, 1 μg/mlmycophenolic acid) was applied.

After approximately two weeks, the clones that appeared were screenedfor antibody production by ELISA. Antibody from a high-producing clonewas prepared by growing the cells to confluency in regular medium (DMEMwith 10% FCS), then replacing the medium with a serum-free medium(Hybridoma SMF; Gibco) and culturing until maximum antibody titers wereachieved in the culture. The culture supernatant was run through aprotein A-Sepharose column (Pharmacia); antibody was eluted with 0.1 MGlycine, 100 mM NaCl, pH 3, neutralized and subsequently exchanged intophosphate-buffered saline (PBS). The purity of the antibody was verifiedby analyzing it on an acrylamide gel, and its concentration wasdetermined by an OD₂₈₀ reading, assuming 1.0 mg of antibody protein hasan OD₂₈₀ reading of 1.4.

Example 4 Affinity of Humanized Anti-B7-2 Antibody

Competitive Binding Assay

The relative affinities, of the murine and humanized 3D1 antibodies forthe B7-2 antigen were determined by competitive binding assays.Three-fold serial dilutions of unlabelled humanized or murine 3D1antibodies were mixed with a fixed amount of radio-iodinated murine 3D1antibody (40,000–50,000 cpm per test in PBS containing 2% fetal calfserum).

1×10⁵ CHO cells expressing cell surface rhB7-2 (CHO/hB7-2) were addedsubsequently and the mixture (in a total volume of 200 ul) was incubatedfor 2 hr at 4° C. with gentle shaking. The cell-antibody suspension wasthen transferred to Sarstedt Micro Tubes (part #72.702) containing 100ul 80% dibutyl phthalate-20% olive oil. After centrifugation in amicrofuge, the Sarstedt tubes were plunged into dry ice for severalminutes. Cell-bound ¹²⁵I was determined by clipping tips of each tube(containing cell pellets) into scintillation vials and counting in agamma counter. Bound and free counts were determined and the ratioplotted against the concentrations of the cold competitor antibodiesaccording to the method of Berzofsky and Berkower (J. A. Berzofsky andI. J. Berkower, in Fundamental Immunology 9ed. W. E. Paul), Raven Press(New York), 595 (1984)).

Cell Line:

Recombinant Chinese Hamster Ovary (CHO) cell lines expressing hB7-2 ontheir membrane surfaces were cloned from cells transfected with B7-2cDNA sequence and G418 resistance. Expression of hB7-2 on the CHO cellsurface over many passages under selective pressure has been confirmedusing murine anti-B7 antibodies and FACS analysis.

Preparation of ¹²⁵I Labeled anti-hB7 mAb and Characterization:

Anti-hB7 antibodies were labeled with ¹²⁵I by reaction with¹²⁵I-Bolton-Hunter reagent according to manufacturers instructions(Amersham Corp. Arlington Hts, Ill.). Protein was separated from freereagent with a NAP-25 column. An HPLC size-exclusion column was used toconfirm that the antibodies remained intact and were not aggregated, andto measure protein concentration against standards prepared fromnon-labeled antibody. Labeling typically resulted in 4 to 8 microcuriesper microgram of protein, or approximately 30 to 60% of the antibodymolecules labeled.

Results:

The competitive binding graph is shown in FIG. 3. Each data pointrepresents the average of triplicate determinations. Results showed thatboth humanized IgG4 and humanized IgG2.M3 anti-human B7-2 antibodieshave a similar high binding affinity as the murine anti-human B7-2antibody (approximately 1×10⁹ M⁻¹), indicating no loss of affinity forB7-2 in the humanization of 3D 1. Both murine and humanized anti-B7-2recognize cell surface expressed hB7-2 with high affinity.

Example 5 Direct Binding of Humanized Anti-B7 mAbs to CHO/hB7 Cells

Cell Binding Assay

Binding assays were begun by plating cells onto 96-well tissue cultureplates at 10,000 CHO/hB7-2 cells per well. Two days later, adherentcells were gently washed with assay buffer containing nonfat dry milkprotein (for blocking nonspecific binding) and sodium azide (to preventinternalization of antibodies by cells). For direct binding assays,¹²⁵I-labeled anti-B7 antibodies (I¹²⁵-murine anti-human B7-2; 826cpm/fmol; humanized anti-human B7-2, 883 cpm/fmol) were serially dilutedin assay buffer and incubated on cells overnight, allowing antibodies tobind to cell-surface B7 and come to equilibrium. Unbound antibody wasgently washed from cells, and bound ¹²⁵I-labeled antibody was detectedusing an ¹²⁵I scintillant and photodetector system. Non-specific bindingto CHO cells was determined for each dilution in the same manner, but oncells expressing the B7-1 molecule that is not recognized by theantibody being tested.

Results:

The direct binding graph is shown in FIG. 4. The data, means oftriplicate wells with nonspecific binding subtracted, were fit to ahyperbolic saturation curve using Graphpad PrismJ software. K_(D) of theantibodies determined as the concentration corresponding to half-maximalbinding indicated that the murine and humanized anti-B7-2 mAbs hadsimilar and high binding affinities (−10⁻⁹m) for B7-2. Both murine andhumanized anti B7-2 antibodies recognize cell surface expressed hB7-2with high affinity.

Example 6 Binding of Humanized Anti-B7 mAbs to Protein Ligands

Affinity Determination by BIACORE

The BIACORE biosensor (BIACORE; Uppsalla, Sweden) was used to determinebinding kinetics of murine and humanized anti-B7-2 human antibodies tohuman hB7-2Ig. HB7-2Ig was immobilized onto the dextran matrix of aBIACORE sensor chip. Humanized and murine anti-human B7-2 were tested at200, 100, 50, and 20 nM. Each dilution was tested 4 times per run and atotal of three separate runs performed. Anti-human B7-2 antibody bindingwas measured in real time by Surface Plasmon Resonance (SPR) and globalanalysis was performed using the bivalent binding model in BIAevaluation software (version 3.1). For each sample, the association(k_(a)), dissociation (k_(d)), and equilibrium dissociation constant(K_(D)) were determined.

Results:

Table 3 reports the mean values obtained for both murine and humanizedanti-human B7-2 mabs. The binding constants for the murine and humanizedanti-B7-2 mAbs determined by SPR shows that the murine and humanizedforms of the anti-B7-2 mAbs are similar and that the murine anti B7-2mAb has a slightly higher binding constant for the immobilized hB7-2 Igthan does the humanized anti-B7-2. The approximately 2.8 fold higheraffinity calculated for the murine anti-B7-2 mAb may represent a real,but slight difference between the murine and humanized anti B7-2 mAbsintroduced during the humanization process. Another possibility may bedue to technical variation in the preparation, processing and analysisof these antibodies. As shown in Examples 4, 5, and 7, a difference wasnot observed in humanized hB7-2 binding affinity in cell based assays.

TABLE 3 Affinity of anti-B7 mAbs as determined by BIAcore mAb K_(D)Humanized K_(D) Murine Anti-B7-2 5.1 × 10⁻⁹M 1.8 × 10⁻⁹MPreparation of hB7-2 Ig:

A soluble form of hB7-2Ig was recovered from culture medium of CHO cellsengineered to secrete this protein. Recombinant hB7-2Ig was derived byfusing the DNA coding sequences corresponding to the extracellulardomain of B7-2 gene to the hinge-CH2-CH3 domains of the human IgG1 heavychain. Recombinant hB7-2Ig was purified from the culture medium byprotein A.

Example 7 Inhibition of T Cell Costimulation by Humanized anti-B7-2

CD28⁺ Tcell/CH0-B7 Proliferation Assay

CD28⁺ T cells, isolated as described herein, were washed once andresuspended in RPMI complete medium, supplemented with 2 ng/ml PMA(Calbiochem), to a cell density of 5×10⁵ cells/ml. The CD28⁺ T cells(100 ul, 5×10⁴ cells) were added to the antibody/CHO/hB7-2 mixture (seebelow), incubated for 3 days at 37° C., 5% CO₂, and T cell proliferationwas measured by pulsing for the last 6 hours of culture with 1 uCi of[³H]-thymidine (NEN, Boston, Mass.). The cells were harvested on afilter and the incorporated radioactivity was measured in ascintillation counter.

Materials:

CD28⁺ T cells were isolated by negative selection with immunoabsorptionfrom human peripheral blood lymphocytes, as described (June et al., Mol.Cell. Biol. 7:4472–4481 (1987)). Buffy coats were obtained byleukophoresis of healthy human donors and peripheral blood lymphocytes(PBL) were isolated by density gradient centrifugation. Monocytes weredepleted from the PBL by plastic absorption. CD28⁺ T cells were isolatedfrom the non-adherent cells by negative selection using antibodies toCD11, CD20, CD16 and CD 14, (this set of antibodies will coat all Bcells, monocytes, large granular lymphocytes, and CD28⁻ T cells) andmagnetic bead separation using goat anti-mouse immunoglobulin-coatedmagnetic particles.

CHO/hB7-2 cells were detached from the tissue culture plates byincubation in phosphate-buffered saline without Ca²⁺ and Mg⁺ (PBS) with0.5 mM EDTA and fixed with freshly prepared paraformaldehyde.

Various concentrations of anti-B7 antibody (in duplicate) werepreincubated for 1 hour at 37° C., 5% CO₂ with 1×10⁴ CHO/hB7-2 cells in100 ul RPMI complete medium (RPMI 1640 medium, 10% fetal bovine serum(FBS),100 U/ml penicillin, 100 ug/ml streptomycin) in a microtiter plate(flat-bottom, 96-well, Costar, Cambridge, Mass.).

Results:

FIG. 5 shows the results of the inhibition of human CD28⁺T cellproliferation by the murine and humanized anti-hB7-2 mAbs. Bothantibodies exhibit dose dependent inhibition of B7-2 driven T cellproliferation with similar IC₅₀ (Inhibitory concentration 50%; amount ofantibody required to inhibit the maximal T cell proliferation by 50%)values of 72 pm (murine anti-hB7-2) and 50 pm (humanized anti-hB7-2)indicating that both antibodies were similar and very effective ininhibiting the B7-2 T cell stimulatory signal. This demonstrates thatthe high affinity anti B7-2 mAbs can block B7-2 functionality byinhibiting (e.g., preventing) the activation and/or proliferation ofhuman T cells. These mAbs are expected to exhibit similar capability inin vivo use to inhibit T cell response.

Example 8 Inhibition of Mixed Lymphocyte Reactions by anti-B7-1 andanti-B7-2 mAbs

Mixed Lymphocyte Reactions (MLR):

Normal peripheral blood lymphocytes (PBL) (responders) were culturedwith irradiated (2,500 cGy) normal donor PBL (stimulators) in RPMI 1640containing 5% heat-inactivated human AB serum at 37° C. in 5% CO₂ at afinal concentration of 10⁶ cells/mL. Where indicated, murine anti-hB7-1or murine anti-hB7-2 antibodies were added alone (10 ug/mL), incombination (10 ug/mL each), and in comparison with CTLA4Ig (10 or 20ug/mL). Cells were cultured in triplicate in microtiter plates in afinal volume of 200 uL and proliferation was assessed by [³H]-thymidineincorporation for the last 16 hours of culture. Secondary MLR wasperformed using the cells derived from the primary MLRs as responders.These cells were washed, cultured overnight, and restimulated as aboveusing the same or different, third party stimulator PBLs. No inhibitorswere added to the secondary MLRs.

Results:

The determinations shown in FIG. 6 were made by performing primaryone-way MLRs in the absence or presence of B7 inhibitors (anti-B7,CTLA4Ig). The proliferation was measured after 3,4, or 5 days ofculture.

In the primary MCR, the additional anti-B7-1 mAb alone had no inhibitoryeffect indicating a minor role for B7-1 alone in driving proliferationof responder T cells. Anti-B7-2 alone inhibited T cell proliferation onall days tested at a level comparable to hCTL4Ig, a recombinant proteinknown to bind to both B7-1 and B7-2. The combination of anti-B7-1 andanti-B7-2 was the most effective inhibitor of T cell proliferation thatcompletely inhibited this response on all days tested. The superiorability of the combined anti-B7-1 and anti-B7-2 to inhibit T cellproliferation, as compared to hCTL4Ig, reflects the higher affinity ofthe anti-B7 mAbs for B7-1 and B7-2 as compared to hCTL4Ig. The combinedanti-B7-1 and anti-B7-2 mAbs were better inhibitors of T cellproliferation than anti-B7-2 alone, demonstrating the need to block bothstimulatory receptors to completely inhibit T cell responses. Theseresults show that complete blockade of the B7-1 and B7-2 costimulatorsmore completely abrogates alloresponsiveness in the MLR. Accordingly,these results indicate that methods of treatment including both antiB7-1 and anti B7-2 antibodies will be even more effective than either ofthe antibodies alone, especially where both costimulatory molecules arefunctional. While the responder/stimulator pair, described herein, wasnot sensitive to inhibition by anti-B7-1 alone, someresponder/stimulator pairs do exhibit moderate (0–50%) anti-B7-1sensitivity.

To determine whether treatment with anti-B7 mAbs in the primary MLR hadresulted in the development of T cell hyporesponsiveness or anergy, theresponder T cells from the primary MLRs were tested in secondary MLRswhere the stimulators were either from the same donor as the primary MLRor from a third party. The results in FIG. 7 show that the responder Tcells from the primary MLR treated with anti-B7-2 alone failed torespond to the same stimulators as used in the primary MLR but retainednormal proliferative response to third party, unrelated stimulatorsindicating that these responder T cells were rendered tolerant to theoriginal stimulator PBLs by treatment with anti-B7-2 and that thetoleraization was specific for the stimulator antigens present in theprimary MLR. With this responder/stimulator pair, treatment withanti-B7-2 alone resulted in tolerance to the stimulator cells; however,with other responder/stimulator pairs, the induction of tolerance maynot be complete.

FIG. 8 shows that the responder T cells from the primary MLR treatedwith anti-B7-1 and anti-B7-2 failed to respond to the same stimulatorsas used in the primary MLR, but retained normal proliferative responseto third party, unrelated stimulators. This indicates that theseresponder T cells were rendered tolerant to the original stimulator PBLsby treatment with the combined anti-B7-1 and anti-B7-2. The resultsobtained with this responder/stimulator pair are typical for otherresponder/stimulator pairs in that tolerance induction is the rule.

Example 9 Inhibition of Immune Responses in Non-Human Primates byAnti-B7 mAbs; Inhibition of Anti-Tetanus Responses

Method:

Twelve tetanus naïve, 4–6 kg male cynomolgus macaques (macacafasicularis) were divided into four experimental groups of three animalper group:

-   -   Group A; received 2 immunizations with 10 Lf Units (Flocculation        Units) i.m. tetanus toxoid on day 0 and 42 (controls).    -   Group B; received 10 mg/kg of each humanized anti-B7.1 (1F1) and        anti-B7.2 (3D1) i.v., at least 90 minutes before 10 Lf units        i.m. tetanus toxoid on day 0; tetanus toxoid immunization only        (without Ab pretreatment) on day 42 (Costimulation blockade with        primary immunization).    -   Group C; received tetanus toxoid immunization only (without Ab        pretreatment) on day 0; 10 mg/kg of each humanized anti-B7.1 and        anti-B7.2 i.v., at least 90 minutes before 10 Lf units i.m.        tetanus toxoid on day 42 (Costimulation blockade with secondary        immunization).    -   Group D; received 10 mg/kg of each humanized anti-B7.1 (1F1) and        anti-B7.2 (3D1) i.v., at least 90 minutes before 10 Lf units        i.m. tetanus toxoid on day 0; received 10 mg/kg of each        humanized anti-B7.1 and anti-B7.2 i.v., at least 90 minutes        before 10 Lf units i.m. tetanus toxoid on day 42 (Costimulation        blockade with primary and secondary immunization).

Serum samples for anti-tetanus antibody testing were collected on days0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, and 84.

Anti-tetanus Antibody ELISA:

96-well ELISA plates were coated with tetanus toxoid lot TP-1002 at 4ug/ml. A four-log titration of serum samples was performed starting at1:100. Ab binding to tetanus was detected with a combination ofmonoclonal anti-human IgG and polyclonal goat anti-rhesus IgMHRP-conjugated antibodies, and developed with TNB substrate.

Results:

FIG. 9 shows the anti-tetanus IgM+IgG responses in monkeys immunizedwith tetanus toxoid and treated with the combined anti-B7-1 andanti-B7-2 mAbs.

Fourteen days after primary immunization, monkeys receiving tetanustoxoid only (Group A) had developed serum anti-tetanus antibody titersof log 3 to 3.5 indicating a normal immune response to the tetanusantigen. An increase anti-tetanus response was seen after the secondtetanus immunization on day 42. Monkeys treated with the combination ofanti-B7.1 and anti-B7.2 before both the primary and secondaryimmunization (Group D) lacked detectable antibody titers until at leastday 84. Monkeys treated with anti-B7.1 and anti-B7.2 before the primaryimmunization only (Group B) maintained an undetectable anti-tetanusantibody titer until at least day 42, and a low (less than log 2.5)until at least day 84. Pretreatment with anti-B7.1 and anti-B7.2 beforesecondary tetanus immunization suppressed the secondary antibodyresponse slightly (Group C vs. Group A). Therefore, the administrationof anti-B7 antibodies concurrent with exposure to a new antigen (tetanusimmunization) can prevent the development of a new antibody response andcan lesson the strength of a secondary response to the same antigen.Since aspects of the rejection of transplanted organs and manyautoimmune diseases involve the generation of antibody responses,treatment with humanized anti-B7 mAbs is useful in preventing organrejection and in treating autoimmune diseases.

Example 10 Serum Half-Life of anti-B7 Antibodies in Non-Human Primates

The murine-anti-hB7-1 and murine-anti-hB7-2 mAbs were tested innon-human primates for serum half-life and target cell saturation. ThreeCynomolgus monkeys were dosed with one dose each of a combination of theanti-hB7-1 and anti-hB7-2 mAbs at 2, 8, or 20 mg each mAb/kg bodyweight. The monkeys were analyzed for mAb binding to PBMC (ProliferativeBlood Mononuclear Cells), serum mAb concentration, and primateanti-mouse antibody (PAMA) response (Table 6). PBMC saturation wasdetermined by flow cytometry (FACS) where PBMCs isolated from the bloodof mAb dosed primates were stained with goat-anti-murine Ig-PE (% invivo) or the PBMC were first reacted with the anti-hB7-1 and anti-hB7-2mAbs followed by detection with the goat-anti-murine Ig-PE (% ex vivo).The level of PBMC saturation at the various time points was calculatedby (% in vivo/% ex vivo)×100. This study shows that PBMC saturation forthe anti-hB7-1 and anti-B7-2 mAbs falls below 80% between days 4 to 6(mAbs @ 2 mg/ks), days 6 to 8 (mAbs @ 8 mg/kg), and days 13 to 20 (mAbs@ 20 mg/kg) depending upon mAb dose. Although not measured directly,there was no apparent dramatic decrease in the numbers of circulatingB7⁺ cells.

Serum half-lives of the anti-hB7-1 and anti-hB7-2 mAbs was measured witha specific ELISA for each mAb using hB7-1Ig or hB7-2Ig as target andgoat-anti-murine Ig HRP/ABTS for detection. These assays were sensitiveto 400 ng/ml and 200 ng/ml for anti-hB7-2 and anti-hB17-1, respectively.PAMA responses were measured using a commercially available kit. Theserum concentrations of the two anti-hB7 mAbs and the PAMA responses areshown at the individual dosage levels for each mAb. Both mAbs exhibitsimilar serum half lines of ˜48 hours as determined at all three dosagelevels. Increasing mAb dosage increased serum mAb concentrations by acomparable factor at all dosages and times tested. When dosed at 20mg/kg, circulating mAb levels of >30 ug/ml were found for each mAb at 6days post dosing.

PAMA responses to the anti-hB7-1 and anti-hB7-2 mAbs were low and werefirst measurable beginning 10 days after serum mAb levels had fallenbelow 10 ug/ml.

The serum half-life of humanized anti-human B7-2 was also determined incynomolgus monkeys (n=6) dosed once with 10 mg/kg of humanized anti B7-2antibody. Serum concentration was monitored by specific ELISA assay foreach antibody using HRP-anti human IgG2 and ABTS.

FIG. 10 shows the serum concentration of the humanized B7-2 mAb incynomolgus monkeys during 42 days after dosing.

The humanized anti-human B7-2 mAb exhibited an extended serum half-lifein cynomolgus monkeys as compared to a value of approximately 2 days forthe murine anti-human B7-2 mAb when dosed at the same level,demonstrating that the humanized anti-human B7-2 mAb was retained incirculation much longer than the murine anti-B7-2 mAb.

TABLE 5 Results from the Preclinical primate studies Dose @ 2 mg eachmAb/kg Dose @ 8 mg mAb/kg Dose @ 20 mg each mAb/kg Time PBL PBL PBLHours Anti-hB7-2 PAMA Saturation Anti-hB7-2 PAMA Saturation Anti-hB7-2PAMA Saturation (Days) ug/mL ng/mL % ug/mL ng/mL % ug/ml ng/ml % 0 BQLNeg. 0 BQL Neg. 0 BQL Neg. 0 .167 61 206 580 .5 59 100 229 25 570 65 152 227 527 3 52 100 230 100 548 100 5 50 139 464 8 44 169 412 24 (1D) 2670 103 100 286 80 48 (2D) 15 100  59 100 196 100 96 (4D) 2.4 75  18 100 83 100 144 (6D) BQL 95 3.9 100  32 100 192 (8D) BQL 65 BQL 100  13 100240 BQL BQL 3.9 (10D) 312 BQL Neg. 5 BQL Neg. 55 BQL Neg. 80 (13D) 4802908 10 4080 10  517 20 (20D) 684 1260 1460 1094 (27D) 816 (34D)

Example 11 Inhibition of Specific T-Cell Responses to Superantigens(Toxic Shock Syndrome Toxin-1; TSST-1)

NODscid mice were populated with human lymphocytes by the administrationof 10e8 human PBLs. After 28 days, the mice were treated with TSST-1 (10mg, I.P.) with or without the treatment with the combined antibodies tohuman B7-1 and B7-2 (500 mg, I.V.). After 14 additional days, thepresence of human lymphocytes, T-cells, and TSST-1 specific T-cells(Vβ2-TCR-cells) in the peritoneal cavity was measured by FACS usingantibodies specific for human CD45, CD4, and human Vβ2-TCR.

TABLE 6 Addition Human T-cells (%) TSST-1 Anti-B7-1 + anti-B7-2 TotalVβ2⁺ − − 10.2 3.9 + − 27.4 12.0 + + 23.4 3.8Results:

Tab le 6 shows the proportion of total human T cells and V_(β)2⁺-TCRhuman T cells (TSST-1 specific) found in the peritoneal cavity ofhu-NODscid mice. Treatment with TSST-1 greatly increased the percentageof human T cells and of TSST-1 specific human T cells (V_(β)2⁺) in thehuNOD-scid mice. Treatment with the anti-human B7-1 and B7-2 mAbsmoderately diminished the total human T cell response and completelyinhibited the expansion of the TSST-1 specific human T cells indicatingthat the anti-B-7 mAbs could effectively inhibit human T cellsuperantigen mediated responses.

The teachings of all the references, patents and/or patent applicationscited herein are incorporated herein by reference in their entirety.

EQUIVALENTS

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims. Those skilled in the artwill recognize or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described specifically herein. Such equivalents are intendedto be encompassed in the scope of the claims.

1. A method for transplanting cells to a patient in need thereof,comprising: a) obtaining cells from a donor, b) obtaining recipientcells from the patient; c) contacting the donor cells with animmunoglobulin specific to B7-1 and an immunoglobulin specific to B7-2,wherein the immunoglobulin specific to B7-2 has a binding affinity of atleast about 10⁷ M⁻¹, and wherein said immunoglobulin comprises anantigen binding region of non-human origin and at least a portion of animmunoglobulin of human origin, further wherein the antigen bindingregion of non-human origin comprises at least one framework regioncontaining a substitution of at least one amino acid to a correspondingamino acid in the III2R heavy chain framework region or the H2F lightchain framework region; d) combining b) and c) to form a mixture, and e)introducing the mixture of step d) to the patient.
 2. The method ofclaim 1, wherein the cells from the donor are derived from bone marrowor blood.
 3. The method of claim 2, wherein the recipient cell is alymphocyte.
 4. The method of claim 3, wherein the immunoglobulinspecific to B7-1, and the immunoglobulin specific to B7-2 are in contactwith the donor cells for a period of time between about 12 hours and 48hours.
 5. The method of claim 4, wherein the period of time is about 36hours.
 6. The method of claim 1, wherein the patient has a disease thatis selected from the group consisting of: a proliferative disease,anemia and myeloid dysplasia syndrome.
 7. The method of claim 6, whereinthe proliferative disease is selected from the group consisting of:leukemia, lymphoma and cancer.
 8. The method of claim 6, wherein theanemia is selected from the group consisting of: sickle-cell anemia,thalassemia and aplastic anemia.
 9. The method of claim 1, wherein theimmunoglobulin specific to B7-1 and the immunoglobulin specific to B7-2are humanized.
 10. The method of claim 1, wherein the immunoglobulinspecific to B7-1 and the immunoglobulin specific to B7-2 are in contactwith the donor cells for a period of time between about 1 hour and 48hours.
 11. The method of claim 1, wherein the immunoglobulin specific toB7-2 can compete with the murine antibody 3D1 (SEQ ID NOS: 2 and 4) forbinding to B7-2.
 12. The method of claim 1, wherein the immunoglobulinspecific to B7-2 is derived from the cell line deposited with the ATCC,Accession No. CRL-12524.
 13. A method for transplanting cells to apatient in need thereof, comprising: a) obtaining cells from a donor, b)obtaining a tissue, an organ, or recipient cells from the patient, c)contacting the donor cells with an immunoglobulin specific to B7-1 andan immunoglobulin specific to B7-2, wherein the immunoglobulin specificto B7-2 has a binding affinity of at least about 10⁷ M⁻¹, and whereinsaid immunoglobulin comprises an antigen binding region of non-humanorigin and at least a portion of an immunoglobulin of human origin,further wherein the antigen binding region of non-human origin comprisesat least one framework region containing a substitution of at least oneamino acid to a corresponding amino acid in the III2R heavy chainframework region or the H2F light chain framework region; d) combiningb) and c) to form a mixture, and e) introducing the mixture of step d)to the patient.
 14. The method of claim 13, wherein the cells derivedfrom the donor are derived from bone marrow, stem cells or immatureblood cells.
 15. The method of claim 13, wherein the immunoglobulinspecific to B7-1 and the immunoglobulin specific to B7-2 are in contactwith the donor cells for a period of time between about 1 hour and 48hours.
 16. The method of claim 13, wherein the immunoglobulin specificto B7-1 and the immunoglobulin specific to B7-2 are humanized.
 17. Themethod of claim 13, wherein the immunoglobulin specific to B7-2 cancompete with the murine antibody 3D1 (SEQ ID NOS: 2 and 4) for bindingto B7-2.
 18. The method of claim 13, wherein the immunoglobulin specificto B7-2 is derived from the cell line deposited with the ATCC, AccessionNo. CRL-12524.